This invention relates to tissue engineering, materials science, cell biology, and plastic surgery.
Over one million surgical procedures in the United States each year involve bone and cartilage replacement (Langer et al., 1993, Science, 920:260-266). The reconstruction of the anatomy of the head and neck presents a considerable challenge because of the unique geometries of facial structures, which require a high degree of complexity and precision in implant fabrication. The use of allografts for these applications is limited by immunological complications, transmission of infectious diseases from the donor, premature resorption of the transplant, and lack of the ability and availability of donor material. Consequently, the use of autologous cartilage and/or bone grafts is considered a primary option. See, e.g., Lovice et al., 1999, Otolaryngol. Clin. N. Am., 32:113-139. However, tissues from locations such as the rib or iliac crest are limited in supply, are associated with significant donor site morbidity, and require significant surgical time to generate an appropriately shaped implant. The use of pre-shaped prosthetic implants made from materials such as polyethylene, silicon, or polytetrafluoroethylene (PTFE) is common, but can be complicated due to higher infection rates and eventual protrusion of implants at the site of the procedure (Cohen et al., 1999, Facial Past. Surg. Clin. N. Am., 7:17-41).
Tissue engineering involves the regeneration of tissues such as bone and cartilage by seeding cells onto a customized biodegradable polymer scaffold to provide a three dimensional environment that promotes matrix production. This structure anchors cells and permits nutrition and gas exchange with the ultimate formation of new tissue in the shape of the polymer material. See, e.g., Vacanti et al., 1994, Transplant. Proc., 26:3309-3310; and Puelacher et al., 1994, Biomaterials, 15:774-778.
The invention is based on the discovery that industrial design and manufacturing techniques, such as injection molding, can be used to create detailed, three-dimensional living tissues.
In general, the invention features methods of making living tissue constructs having a specific, e.g., predetermined shape by providing a negative mold having a predetermined, three-dimensional shape; suspending isolated tissue precursor cells in a hydrogel to form a liquid hydrogel-precursor cell composition; introducing the liquid hydrogel-precursor cell composition into the mold; inducing, e.g., controllably inducing, gel formation to solidify the liquid hydrogel-precursor cell composition to form a living tissue construct; and removing the living tissue construct from the mold after gel formation. For example, the cells can be epidermal cells, chondrocytes and other cells that form cartilage, macrophages, adipocytes, dermal cells, muscle cells, hair follicles, fibroblasts, organ cells, osteoblasts, osteocytes and other cells that form bone, endothelial cells, mucosal cells, pleural cells, ear canal cells, tympanic membrane cells, peritoneal cells, Schwann cells, corneal epithelial cells, gingiva cells, central nervous system neural stem cells, or tracheal epithelial cells.
The hydrogels can be alginate (e.g., at a concentration of 0.5% to 8% or 1% to 4%, e.g., 2%), chitosan, pluronic, collagen, or agarose. The hydrogels can also be polysaccharides, proteins, polyphosphazenes, poly(oxyethylene)-poly(oxypropylene) block polymers, poly(oxyethylene)-poly(oxypropylene) block polymers of ethylene diamine, poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acid and methacrylic acid, poly(vinyl acetate), or sulfonated polymers. In these methods and depending on the hydrogel, gel formation can be induced by contacting the liquid hydrogel with a suitable concentration of a divalent cation, such as Ca++, e.g., at a concentration of 0.2 g/ml of an alginate solution.
Once the construct is made, it can be directly implanted, or cultured, e.g., in vitro, to allow the cells to grow within the hydrogel construct, e.g., for a period of 1 to 30 days.
In another aspect, the invention features methods of reconstructing an anatomical feature in a mammal by providing a suitable negative mold having a three-dimensional negative shape of the anatomical feature; suspending isolated tissue precursor cells in a hydrogel to form a liquid hydrogel-precursor cell composition; introducing the liquid hydrogel-precursor cell composition into the mold; inducing gel formation to solidify the liquid hydrogel-precursor cell composition to form a living tissue construct; removing the tissue construct from the mold after gel formation; and implanting the tissue construct into the mammal. Alternatively, the method can include obtaining a living tissue construct having the three-dimensional shape of the anatomical feature; and implanting the tissue construct into the mammal. In this method, the construct can be prepared by the new methods described herein.
The invention also features the injection-molded living tissue constructs made by the new methods. These constructs can have a variety of shapes, e.g., they can be in the shape of particular cartilage adjacent a joint, a bone, a portion of a bone, or a bone defect.
A xe2x80x9chydrogelxe2x80x9d is a substance formed when an organic polymer (natural or synthetic) is set or solidified to create a three-dimensional open-lattice structure that entraps molecules of water or other solution to form a gel. The solidification can occur, e.g., by aggregation, coagulation, hydrophobic interactions, or cross-linking. The hydrogels employed in this invention rapidly solidify to keep the cells evenly suspended within a mold until the gel solidifies. The hydrogels are also biocompatible, e.g., not toxic, to cells suspended in the hydrogel.
A xe2x80x9chydrogel-cell compositionxe2x80x9d is a suspension of a hydrogel containing desired tissue precursor cells. These cells can be isolated directly from a tissue source or can be obtained from a cell culture. A xe2x80x9ctissuexe2x80x9d is a collection or aggregation of particular cells embedded within its natural matrix, wherein the natural matrix is produced by the particular living cells. A xe2x80x9cliving tissue constructxe2x80x9d is a collection of living cells that have a defined shape and structure. To be xe2x80x9cliving,xe2x80x9d the cells must at least have a capacity for metabolism, but need not be able to grow or reproduce in all embodiments. Of course, a living tissue construct can also include, and in some embodiments preferably includes, cells that grow and/or reproduce.
xe2x80x9cTissue precursor cellsxe2x80x9d are cells that form the basis of new tissue. Tissue cells can be xe2x80x9corgan cells,xe2x80x9d which include hepatocytes, islet cells, cells of intestinal origin, muscle cells, heart cells, cartilage cells, bone cells, kidney cells, cells of hair follicles, cells from the vitreous humor in the eyes, cells from the brain, and other cells acting primarily to synthesize and secret, or to metabolize materials. In some embodiments, these cells can be fully mature and differentiated cells. In addition, tissue precursor cells can be so-called xe2x80x9cstemxe2x80x9d cells or xe2x80x9cprogenitorxe2x80x9d cells that are partially differentiated or undifferentiated precursor cells that can form a number of different types of specific cells under different ambient conditions, and that multiply and/or differentiate to form a new tissue.
An xe2x80x9cisolatedxe2x80x9d tissue precursor cell, such as an isolated nerve cell, or an isolated nerve stem or progenitor cell or bone cell, or bone stem or progenitor cell, is a cell that has been removed from its natural environment in a tissue within an animal, and cultured in vitro, at least temporarily. The term covers single isolated cells, as well as cultures of xe2x80x9cisolatedxe2x80x9d stem cells, that have been significantly enriched for the stem or progenitor cells with few or no differentiated cells.
As used herein, xe2x80x9cnegative moldxe2x80x9d means a concave mold into which a liquid can be introduced for subsequent solidification. The mold is xe2x80x9cnegativexe2x80x9d in the sense that concavity of the mold represents convexity in the object to be formed.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, useful methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflicting subject matter, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The invention has many advantages. For example, the new methods reduce the number of manufacturing steps needed to prepare precise, three-dimensional biological tissues. The new methods also provide increased uniformity of cell seeding throughout the construct, and increased efficiency of cell containment within the construct.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.